An optimization of supplements and physical factors for growth of hemocytes culture from Penaeus vannamei (White shrimp) in selective medium.

BACKGROUND: Standardization of cell culture medium plays a vital role in the development of primary or continuous cell line. Apart from the basal media, supplements in the medium and various physical factors promote the cell growth. With this context, the study was carried out to optimize the culture medium using various supplements and physical factors for the growth of hemocytes culture from Penaeus vannamei. METHODS: Various concentrations of Fetal Bovine Serum (FBS; 1-25%), Shrimp Muscle Extract (SME; 1-25%) and basic Fibroblast Growth Factor (bFGF; 0.5-5 ng mL -1) were attempted to optimize the cell culture media for the development of primary hemocytes culture of P. vannamei. Various pH, temperature and osmolality was also screened to optimize the medium. RESULTS: 15% FBS was ideal for the healthy morphology of cells with rapid replication. SME supplementation at 5-20% supported the cell growth for 24 h but only 30% of cell viability was observed after 48 h. bFGF (0.5-5 ng mL-1) enhanced cell growth in the medium with 15% FBS; The ideal pH level was examined by preparing the HBSCM-5 medium at pH between 6.8-8.0. Osmolality of 730 ± 20, pH of 7.2 and temperature of 28 °C resulted in the healthy cells with good morphology. NSW supplement supported the cell growth at low concentrations of salt; however, more than 2% salt concentrations cells did not form fibroblast-like morphology and instead a crystal-like morphology was observed. CONCLUSION: The hemocytes culture were optimized for use as an in vitro cell culture system by testing cell growth on HBSCM-5 medium with various supplements, growth factors and physical parameters.

Medium optimization and characterization of cell culture system from Penaeus vannamei for adaptation of white spot syndrome virus (WSSV).

The lack of shrimp cell lines and difficulty in establishing shrimp cell culture systems, with an appropriate medium is a major concern in the aquaculture sector. The present study attempts to address this issue by developing an in vitro cell culture system from various tissues (hemocytes, heart, lymphoid tissue, hepatopancreas, gill, eye stalk, and muscle) of Penaeus vannamei (P.vannamei) using commercially available L-15 medium. The cell culture medium was formulated using five different media such as HBSCM-1, HBSCM-2, HBSCM-3, HBSCM-4, and HBSCM-5 containing L-proline and glucose with fetal bovine serum (FBS) supplements. Among the different media used, the HBSCM-5 medium with supplements showed good attachment and proliferation of cells with fibroblast-like, epithelioid, round, and adherent cell morphology in hemocyte culture. The same medium was further screened using different tissues to enhance the cell growth. The hemocytes, heart, and lymphoid tissue cells were passaged five times and maintained up to 20 days. Hepatopancreas and gill cells initially showed good morphological features and survived for more than ten days following subculture cells. Eye stalks and muscle cells perished within five days and did not show any unique morphology. The primary hemocyte cells were subjected to species identification, using cytochrome oxidase subunit I (COI) gene. To assess the primary hemocyte cell culture, cells were used for in vitro propagation of white spot syndrome virus (WSSV) and confirmed by the conventional polymerase chain reaction (PCR). Similarly, the primary cells were treated with bacterial extracellular products (ECPs) from Vibrio parahaemolyticus and Vibrio harveyi, to evaluate the cytotoxicity.

Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India.

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15 µm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99-100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.

Loose shell syndrome of farmed Penaeus monodon in India is caused by a filterable agent.

Loose shell syndrome (LSS) of farmed black tiger shrimp Penaeus monodon has been reported from Indian shrimp farms since 1998 and is recognized as a major disease problem causing significant economic loss to the shrimp aquaculture sector. Unlike the rapid mortalities associated with viral pathogens such as white spot syndrome virus and yellow head virus, progression of LSS is gradual, leading to low-level progressive mortalities. The signs of LSS include a flaccid spongy abdomen due to muscular dystrophy, space between the exoskeleton and muscle, and a shrunken hepatopancreas. The feed conversion efficiency is reduced, and shrimp have poor meat quality, caused by impairment of the hepatopancreatic functions such as digestion and absorption as evidenced by the atrophy of the hepatopancreas. Histopathological investigations on LSS-affected shrimp showed shrinkage of extensor and flexor muscles with occasional hemocytic infiltration. The hepatopancreas showed inflammation of hepatopancreatic tubules with enlargement of intertubular spaces, hemocytic infiltration, and low levels of lipid reserves in the R cells. In advanced stages of LSS, many tubules were in highly necrotic condition with a sloughed epithelium, reflecting the dysfunction of the digestive gland. LSS could be induced in healthy tiger shrimp by challenge studies using membrane-filtered LSS-affected shrimp tissues, suggesting involvement of a filterable infectious agent.

Anti-oxidant status in embryonic, post-hatch and larval stages of Asian seabass (Lates calcarifer).

The concentrations of anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and selenium-dependent glutathione peroxidase (SeGPx), and low molecular weight free-radical scavengers such as reduced glutathione (GSH) and ascorbic acid (vitamin C) were evaluated during the period from gastrulation (GS) to 25 days post-hatch (dph) in the larvae of Asian Seabass, Lates calcarifer. Oxidative damage due to lipid peroxidation (LPO) was also assessed, by evaluation of the formation of malondialdehyde (MDA). All the three anti-oxidant enzymes, SOD, CAT and GPx, showed high activities during gastrulation, suggesting an increased metabolic rate during the period of embryonic development. Though the SOD activity apparently decreased progressively during 3-20 dph of larval development, the difference was not significant. CAT showed high activity during gastrulation and remained constant up to 3 dph, suggesting an increased need to metabolise hydrogen peroxide (H2O2) and organic peroxides. In contrast, SeGPx activity increased progressively from 5 dph to 25 dph during larval development, indicating an increased need to detoxify lipid peroxides. This is evident from the observation of increased lipid peroxidation from 10 dph to 25 dph during larval development. GSH levels were low at gastrulation, indicating increased metabolic rate and formation of lipid radicals during this period, corresponding to the decrease in the level of ascorbic acid, which is consumed for regeneration of GSH.

Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus.

AIMS: To identify the causative agent of the mortality in the fish, Mugil cephalus, in Muttukadu lagoon. METHODS AND RESULTS: An enteric bacterium from the kidneys of moribund fish M. cephalus, was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis. CONCLUSIONS: The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus, found in Muttukadu lagoon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae, harbouring cationic factor, between human and fish may be of human health importance.

Phenotypic and molecular typing of Vibrio harveyi isolates and their pathogenicity to tiger shrimp larvae.

AIMS: The objective of the present study was to identify the biotype(s) and molecular type(s) of Vibrio harveyi associated with pathogenicity in tiger shrimp (Penaeus monodon) larvae. METHODS AND RESULTS: Five luminescent and four nonluminescent V. harveyi isolates were subjected to phenotyping and random amplified polymorphic DNA (RAPD) fingerprinting, and pathogenicity testing to P. monodon mysis. Four isolates induced 34-41% mortality of P. monodon mysis when challenged at the rate of 10(6) CFU ml(-1) within 60 h. Sucrose-fermenting biotypes of V. harveyi appeared to be associated with pathogenicity to larval shrimp. Higher temperature and salinity appeared to play a role on the onset of vibriosis and mortality in the challenged larval shrimp. Pathogenic isolates of V. harveyi could be demarcated as revealed by their clustering in the dendrogram constructed based on the RAPD fingerprints. CONCLUSIONS: Nonluminescent V. harveyi also appear to be important aetiological agents of vibriosis of shrimp larvae. Sucrose-fermenting biotypes are likely to be pathogenic. High temperature may trigger onset of vibriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Biotyping of V. harveyi isolates and looking for traits, such as ability to ferment sucrose may be helpful in identifying the pathogenic forms, and such approach requires to be investigated further with larger number of isolates.